1. Field of the Invention
The present invention relates to a new homogeneous cytosolic binding protein (entitled "FKBP12.6") which binds FK-506 but not cyclosporine A, is unstable to heating at 56.degree. C. for 30 minutes, and has a molecular weight of about 10-12 kilodaltons. The FKBP12.6 protein has peptidyl-proline isomerase enzymatic activity. Moreover, FKBP12.6 binds to and inhibits the phosphatase calcineurin in the presence of FK-506.
2. Brief Description of Disclosures in the Art
Immunoregulatory abnormalities have been shown to exist in a wide variety of "autoimmune" and chronic inflammatory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type 1 diabetes mellitus, type 2 adult onset diabetes, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, and Graves ophthalmopathy. Although the underlying pathogenesis of each of these conditions may be quite different, they have in common the appearance of a variety of autoantibodies and self-reactive lymphocytes. Such self-reactivity may be due, in pan, to a loss of the homeostatic controls under which the normal immune system operates.
Similarly, following a bone-marrow or an organ transplantation, the host lymphocytes recognize the foreign tissue antigens and begin to produce antibodies which lead to graft rejection.
Cyclosporin A which was approved by the US FDA in 1983 is currently the leading drug used to prevent rejection of transplanted organs. The drug acts by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein. Although cyclosporin A is effective in fighting transplant rejection, it is nephrotoxic and is known to cause several undesirable side effects including kidney failure, abnormal liver function and gastrointestinal discomfort.
Fujisawa United States, European and Japanese patents and applications (U.S. Pat. No. 4,894,366, EPO Publication No. 0,184,162 and PBJ Disclosure 63-17884) and publications (J. Am. Chem. Soc., 109, 5031 (1987), and J. Antibiotics, 40, 1249 (1987)) disclose "FK-506", 17-allyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[2 2.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone, FR-900506, tacrolimus, which is reputed to be 100 times more potent than cyclosporine. The macrolide is produced by fermentation of a particular strain of Streptomyces tsukubaensis.
Extensive clinical studies with FK-506 in the treatment of resistance to organ transplantation have been conducted (see e.g., G. L. Bumgardner, et al., Gastroenterol. Clin., 22 (2), 421-449 (1993)). In addition, FK-506 and related compounds have been suggested to be useful in the treatment of obstructive airways disease, particularly asthma (PCT Publication WO 90/14826), male pattern alopecia or alopecia senilis (EPO Publication No, 0,423,714), rheumatoid arthritis (C. Arita, et al., Clincial exp. Immunol., 1990, 82, 456-461; N. Inamura, et al., Clin. Immunol. Immunopathol. 1988, 46, 82-90), recent-onset diabetes (N. Murase, et al., Diabetes, 1990, 39, 1584-86; N. Murase, et al., Lancet, 1990, 336, 373-74), posterior uveitis (H. Kawashima, Invest, Ophthalmol. Vis. Sci., 1988, 29, 1265-71), hepatic injury associated with ischemia (M. Sakr, et al., Life Sci., 1990, 47, 687-91) allergic encephalomyelitis (K, Deguchi, et al., Brain Nerve, 1990, 42, 391-97), glomerulonephritis (J. McCauley, et al., Lancet, 1990, 335,674), systemic lupus erythematosus (K. Takabayashi, et al., Clin. Immunol. Immunopathol., 1989, 51, 110-117), multidrag resistance (M. Naito, et al., Cancer Chemother. Pharmacol., 1992, 29, 195-200), inflammation of mucosa and blood vessels (PCT Publication WO 92/17754), cytomegalovirus infection (UK Publication GB 2,247,620A), idiopathic thrombocytopenic purpura and Basedow's disease (PCT Publication WO 91/19495). Also, these compounds are reputed to possess topical activity in the treatment of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically-mediated illnesses (EPO Pub. No. 0,315,978).
Although FK-506 has been employed for the treatment of some of the above disorders and diseases, neurological toxicities, which include drowsiness, lethargy, tremors, and aggressiveness (Ohara, K., et al., Transplant. Proc., 22, 83-86 (1990); K. Kumano, et al., Transplant. Proc., 23, 512-515 (1991)), limit its broader use. Accordingly, it would be desirable to understand the mechanism by which FK-506 exerts its immunosuppressive and its toxic effects, as well as develop assays to measure its concentration in biological fluids.
FK-506 shares a number of immunosuppressive properties with cyclosporine A, although it is 10-100 times more potent in this regard. These similarities suggest that both agents may share a similar mechanism of action at the biochemical level (see S. Lin, et al., Cellular Immunology, 133,269-284 (1991)). For example, Cyclosporine A is known to bind to the cytosolic protein, cyclophilin (R. E. Handschumacher, et al., Science, 226, 544-546 (1984)). The cyclophilin.cndot.cyclospofine A complex binds to and inhibits the phosphatase calcineurin (CAN) which is required for T-cell activation and immune system function (S. O'Keefe, et al., Nature, 357, 692-694 (1992)). Cyclophilin has also been shown to possess an enzymatic activity, which catalyzes the cis-trans isomerization of peptidyl prolyl bonds (N. Takahashi et al., Nature, 337, 473-475; and Fischer et al., Nature, 337,476-478).
FK-506 binds to an abundant, cytosolic 11.8 kDa protein termed the FK-506 binding protein (FKBP12) that is expressed universally among tissues and throughout eukaryotic phylogeny (J. Siekierka, et al., Nature, 341,755-757 (1989); M. Harding, et al., Nature, 341,758-760 (1989); U.S. Pat. No. 5,109,112). Like the cyclophilin.cyclosporin A comples, the FKBP12.FK-506 complex inhibits calcineurin, a calcium-dependent, serine-threonine phosphatase that is a vital component of the signal transduction pathway resulting in early lymphokine gene transcription (J. Liu, et al., Cell, 66, 807-815 (1991); J. Friedman, et al., Cell, 66, 799-806 (1991); N. Clipstone, et al., Nature, 357, 695-697 (1992); S. O'Keefe, et al., Nature, 357,692-694 (1992)). Human FKBP12 has been cloned (N. Maki, et al., Proc. Natl. Acad. Sci., 87, 5440-5443 (1990); R. Standaert, et al., Nature, 346, 671-674 (1990); G. Wiederrecht, et al., J. Biol. Chem., 267(21), 753-760 (1992)) and the amino acid sequence of the bovine FKBP12 has been reported (N. Mozier, et al., Eur. J. Biochem., 194, 19-23 (1990); J. Siekierka, et al., J. Biol. Chem., 265, 21011-21015 (1990)).
FKBP12 is not a lymphoid-specific protein, but is widely distributed in tissues and throughout the phyla. The FKBP12 protein is a member of a new class of enzymes, collectively termed peptidylprolyl isomerases (PPIases) which catalyze isomerization between the cis and trans forms of the Xaa-Pro bond in peptides and proteins. Known FK-506 binding proteins (FKBPs) include: FKBP12 (U.S. Pat. No. 5,109,112); FKBP13 (Proc. Natl. Acad. Sci., 88, 6677-6681 (1991)); FKBP25 (G. Wiedderecht, et al., Biochem. Biophys. Res. Commun., 185, 298-303 (1992)); and FKBP52 (G. Wiedderecht, et al., J. Biol. Chem., 267, 21753-21760 (1992) (see generally, A. Galat, FEBS, 216, 689-707 (1993)). Their ubiquity and abundance suggest that FKBPs have an important role in cellular physiology, perhaps in accelerating interconversion between rotational conformers of denatured or recently synthesized proteins. Unlike FKBP12, however, the other reported FKBPs do not bind calcineurin when complexed with FK-506. Accordingly, the isolation and identification of other FKBPs would help to delineate the FK-506 mechanism of therapeutic action and toxicity in the cell.